LOCUS       sequence-260183-        2987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro expression of E. coli MBP with C terminal 4xDQNAT 
            glycosylation sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2987)
  AUTHORS   Stark JC, Jaroentomeechai T, Moeller TD, Hershewe JM, Warfel KF, 
            Moricz BS, Martini AM, Dubner RS, Hsu KJ, Stevenson TC, Jones BD, 
            DeLisa MP, Jewett MC
  TITLE     On-demand biomanufacturing of protective conjugate vaccines.
  JOURNAL   Sci Adv. 2021 Feb 3;7(6). pii: 7/6/eabe9444. doi: 
            10.1126/sciadv.abe9444. Print 2021 Feb.
  PUBMED    33536221
REFERENCE   2  (bases 1 to 2987)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2021-02-3"; volume: "7(6). pii: 7/6/eabe9444. doi"; pages: " 
            10.1126/sciadv.abe9444. Print 2021 Feb"
COMMENT     Sequence Label: pJL1-MBP-4xDQNAT
FEATURES             Location/Qualifiers
     source          1..2987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     34..53
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        34..52
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             84..106
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             114..1214
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGTVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1071..1080
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1188..1211
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1317..1334
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1389..1407)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1403..1450
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1468..2283)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2191..2210
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2275..2294
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(2405..2987,1..6)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2894..2913
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tggaaaaacg ccagcaacgc gatcccgcga aattaatacg actcactata gggagaccac
       61 aacggtttcc ctctagaaat aattttgttt aactttaaga aggagatata catatgaaaa
      121 tcgaagaagg taaactggta atctggatta acggcgataa aggctataac ggtctcgctg
      181 aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata
      241 aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac attatcttct
      301 gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg
      361 acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca
      421 agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc
      481 tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga
      541 aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg
      601 ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg
      661 gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca
      721 aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa
      781 cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt
      841 atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc
      901 tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcgaaagag ttcctcgaaa
      961 actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtaccg
     1021 tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccaccatgg
     1081 aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg
     1141 ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga
     1201 aagacgcgca gactcgtatc accaagctcg aggatcagaa cgcgaccggc ggtgaccaaa
     1261 atgccacagg tggcgatcaa aacgccaccg gcggtgacca gaatgcgaca gtcgaccatc
     1321 accatcatca ccattaagtc gaccggctgc taacaaagcc cgaaaggaag ctgagttggc
     1381 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     1441 gggttttttg ctgaaagcca attctgatta gaaaaactca tcgagcatca aatgaaactg
     1501 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     1561 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
     1621 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     1681 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gcttatgcat
     1741 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     1801 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
     1861 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     1921 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg
     1981 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     2041 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     2101 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
     2161 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     2221 agcatccatg ttggaattta atcgcggctt cgagcaagac gtttcccgtt gaatatggct
     2281 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat
     2341 atttttatct tgtgcaatgt aacatcagag attttgagac acaacgtgag atcaaaggat
     2401 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     2461 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     2521 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     2581 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     2641 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2701 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2761 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2821 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2881 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2941 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagccta
//
